1. Technical Field of the Invention
The present invention relates to a reagent composition and method for identification of reticulated cells from other cell types in a blood cell sample by using optical measurement. More particularly, the reagent composition comprises a nucleic acid dye and sphering agent. More particularly, the method enables the identification of reticulated platelets and reticulated erythrocytes. In addition, the method provides for a concurrent determination of cell by cell hemoglobin in the blood cell sample by suitable electrical and optical measurements.
2. Discussion of the Prior Art
Reticulated cells are defined as being any blood cell which contains reticulum. Reticulated cells include reticulated erythrocytes and reticulated platelets. Reticulated erythrocytes are commonly known as reticulocytes.
The identification of reticulated cells has focused on reticulocyte analysis. Prior art in reticulocyte enumeration methods suitable for automated hematology is based on reagents containing acridine orange (AO), oxazine 750, thiazole orange (TO) and its derivatives and coriphosphine-O (CPO). Acridine orange, thiazole orange and coriphosphine-O are all brightly fluorescent when bound to RNA. Each of these dyes are cell membrane permeant, i.e. they transport through the membrane of the reticulocyte cells freely. This makes them attractive and useful for automated hematology application. However, each of these reagents present their own methods of analysis and problems.
Specific examples using acridine orange (AO) are cited in U.S. Pat. Nos. 5,075,556; 5,360,739 and 5,411,891 each to Fan et al.; and cited in publications to Vander et. al., (1993) J. Lab. Clin. Med, 62:132, Thaer et. el. (1970) "Microfluorometric analysis of the reticulocyte population in peripheral blood of mammals" in Cytology Automation, DMD Evans (ed.), E&S Livingistone, Edinburgh (1970), pp. 180-195; and Seligman et. al., (1983) American J. Hematol., 14:57-66].
Specific examples using oxazine 750 are shown in U.S. Pat. Nos. 5,284,771 and 5,633,167 each to Fan et al.; and oxazine 750 or new methylene blue shown in U.S. Pat. Nos. 5,350,695 and 5,438,003 each to Colella et al.
Specific examples using thiazole orange (TO) are shown in U.S. Pat. Nos. 4,883,867 and 4,957,870; and cited in publications Cytometry (1986) 7:508 L. G. Lee et. al.; Van Hove et. al. (1990) Clin. Lab. Hemat., 12:287-299; Carter et. al. (1989) Clin. Lab. Haemat, 11:267-271].
Specific examples using coriphosphine-O (CPO) are shown in U.S. Pat. No. 5,639,666 to Shenkin.
In addition, other reagents and automated methods have been developed for analysis of reticulocytes. U.S. Pat. No. 4,985,174 to Kuroda et al. disclose a reagent containing auramine-O for the fluorescent staining of reticulocytes in a sample of whole blood to permit determination of reticulocytes by means of a flow cytometer. U.S. Pat. Nos. 5,773,299 and 5,691,204 each to Kim et al. disclose an unsymmetrical cyanine dye.
Another form of reticulated cells are reticulated platelets. Reticulated platelets are released into the peripheral blood following megakaryocytic fragmentation. Newly formed platelets, although anucleated, contain some rough endoplamic reticulum and messenger RNA (mRNA) and are also able to synthesize small amounts of protein. Their measurement has considerable clinical utility for monitoring thrombopoiesis and platelet turnover as the mRNA is unstable and degrades within twenty four hours in animal models.
Prior art in reticulated platelet analysis has been based on thiazole orange as shown in Robinson et al. (1998) "Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non-specific labeling of dense granules by fluorescent dyes" in British Journal of Haematology, 100, pp. 351-357 and Richards, et al. (1996) "Measurement of reticulated platelets following peripheral blood progenitor cell and bone marrow transplantation: implications for marrow reconstitution and the use of thrombopoietin" Bone Marrow Transplantation, 17 pp. 1029-1033. In addition auramine-O has been used in Watanabe et al. (1995) "Automated measurement of reticulated platelets in estimating thrombopoiesis" Eur. J. Haematol 54 pp. 163-171. Both dyes are a fluorescent nucleic acid dye excitable at 488 nm and are suitable for argon lasers in standard flow cytometers.
Prior to the invention disclosed herein, prior art determinations of reticulated platelets did not conform to any standardized results or methodologies. Fundamentally, each laboratory set its own standard of practice and measurement system. Prior analysis led to the conclusion among some laboratories, but not all, that reticulated platelets constituted approximately 2% to 20% of a normal platelet population.
Thus, there exist a need for a reagent composition with specificity and method that enables an accurate determination of reticulated cells in less than 60 seconds. In addition, the reagent and method finds particular utility for the automated method for the identification of reticulated platelets.